The purpose of this research is to investigate the molecular mechanisms of action of biologically active proteins from arthropod disease vectors and pathogenic microorganisms. We use biological and physical techniques to characterize and understand the modes of action of pharmacologically active components from the saliva of blood-feeding vector insects and ticks, as well as immunomodulatory components secreted by parasitic organisms such as Toxoplasma and Schistosoma. Proteins and small molecules found in the saliva of vectors inhibit the host hemostatic responses and are essential for the successful completion of a blood meal. Most vector borne diseases are transmitted during feeding, so elucidation of the physiology and biochemistry of this process is necessary for understanding disease transmission. Saliva has also been shown to have pronounced effects on host inflammatory and immune responses which persist after feeding and can dramatically alter the environment for the pathogen after transmission. Determining the specific role of salivary molecules in these processes is essential for the understanding their importance to pathogen survival after transmission Over the past several years we have identified the functions of numerous salivary molecules involved primarily in overcoming host hemostatic defenses. The raw material for these studies comes from the analyses of salivary transcriptomes produced in collaboration with Dr. Jose Ribeiro. Bioinformatic analysis of sequence data is used to predict function of salivary proteins. Candidate proteins are then expressed in bacterial or eukaryotic cell systems. The proteins are purified and assayed using a variety of methods. Functionally characterized proteins are then produced in larger quantity for structural and other biophysical studies. During the 2017 fiscal year we have 1) Characterized and determined the crystal structure of a protein circulates in the blood of mosquitoes and binds juvenile hormone, a hormone essential for development and reproduction. This work has been published 2) Determined the molecular mechanism of action of LJL143, an inhibitor of the alternative pathway of complement in the sand fly. 3) Determined the crystal structure of albicin, an inhibitor of the alternative pathway of complement from the mosquito Anopheles albimanus. 4)Initiated a project to determine the structure of the albicin-C3bBb complex by cryo electron microscopy. 5) Completed a collaboration with Dr. Bruno Arca in Rome on characterization of the thrombin inhibitor of Anopheles gambiae. 6) Assisted Dr. Wellems and Dr. Mu from the LMVR in the development of diagnostic methods for Plasmodium infection, by determining the affinities of antigen-antibody complexes using surface plasmon resonance. 1) Over the past decade we have shown that the D7 protein family in mosquito saliva functions by sequestering host-produced mediators of hemostasis and inflammation. Among these are the eicosanoids thromboxane A2 and leukotriene C4. Since these mediators are not known to function in the mosquito, the D7 proteins must be derived from ancestors with different function. I have identified mJHBP, a protein that shows sequence conservation between various genera of mosquitoes and is similar to salivary D7s but has changes in amino acid residues important for binding of vertebrate eicosanoid ligands. Analysis of various tissues and life stages showed that this protein is expressed in the fat body and directed to the blood of the insect. Moreover, the protein is found in adult males and females. Analysis of mJHBP ligand binding using calorimetry showed that the protein binds the important insect hormone, juvenile hormone, with high selectivity. This hormone is essential for metamorphosis, egg development and male mating behavior. We have crystallized the protein, and determined its structure in the presence of bound juvenile hormone III. The ligand is stabilized by relatively large conformational change, and the binding pocket is highly selective for the natural 10R enantiomer of the hormone. This work has recently been published in The Journal of Biological Chemistry. We are continuing to study the binding properties of the protein and to evaluate the effect gene knockdown using double stranded RNA methods. 2) Inhibition of the complement cascade is an important feature of saliva from blood feeding vectors. Activation of the complement system in host blood can result in the destruction of insect tissues and production of proinflammatory anaphylatoxins. We have identified lufaxin, an inhibitor of the alternative pathway of complement in the saliva of the sand fly Lutzomyia longipalpis. Using surface plasmon resonance and reconstituted enzymatic systems, we have found that lufaxin specifically binds to the C3bB complex, and prevents its activation (cleavage) to C3bBb by factor D. It appears that the protein recognizes an open conformation of factor B that is recognized by the factor D protease. Inhibitors that bind to C3bBb, the mature form of the C3 convertase, have been described previously, but this is the first inhibitor described that specifically blocks the formation of the convertase. 3) In a project related to topic 2, we have determined the crystal structure of albicin, a previously characterized inhibitor of the alternative pathway of complement that binds to the activated C3 convertase, C3bBb. This inhibitor belongs to the SG7 family of mosquito salivary proteins that occurs throughout the anopheline subfamily of mosquitoes. Anti complement activity is not present in the saliva of the Plasmodium vector species of Africa and Southeast Asia, even though they contain members of the SG7 family. This structure will allow us to understand the molecular determinants of anti complement activity, as well as the functions of SG7 forms that do not inhibit complement. 4) In order to understand the mechanism of albicin action we working to determine its structure in complex with C3bB using cryo EM. I a collaboration with Dr. Beth Fischer from the RML we have developed a protocol for preparation of C3bBb-albicin bound to the stabilizing molecule properdin. Currently we are working to obtain negative stained images, that may be sufficient for developing a working model of the complex. All of the individual components have been structurally characterized using crystallography (mainly by the group of Piet Gros), and the native structure of C3bBb has also been determined (also by the Gros group). 5)The saliva of anopheles mosquitoes contains an extremely potent anti coagulant that targets thrombin, the ultimate protease of the coagulation cascade. During the past year, I collaborated with Dr. Bruno Arca of the University of Rome, to characterize the variant of this peptide occurring in the saliva of Anopheles gambiae. The peptide contains an RGD motif that is associated with integrin inhibitors, suggesting that it may block platelet activation. We found that the inhibitor did not inhibit platelet activation, indicating that it does not block the fibrinogen receptor alphaIIbbeta3. This study has been recently published in the Journal of Biological Chemistry. 6) Finally, I worked to provide support for a project from Dr. Wellems lab aimed at developing a very sensitive test for blood stage malaria. Binding information for the interaction of a set of monoclonal antibodies with their target antigens was required, and I obtained this data in collaboration with Dr. Mu using surface plasmon resonance. A paper describing this work in In Press in the Journal of Infectious Diseases.